Gene expression in bacteria, as in any organism, requires that a promoter be present in the regulatory region located 5′ (i.e. upstream) from the coding sequence in order to direct the gene's transcription. Promoters are classified as constitutive promoters and regulated promoters. In commercially useful bacterial expression systems, regulated promoters have proven particularly useful because they permit increase in the organismal biomass while a desired gene(s) is inactive. This allows the host organism to devote maximal energy to cell division and growth. When the regulated promoter is then activated/induced, more cells will be available to express the desired gene(s), thereby increasing the yield of the desired gene product(s).
Regulated promoters include: (1) activatable promoters, i.e. promoters that are inactive until an activator protein binds to the 5′ regulatory region; and (2) repressible promoters, i.e. promoters that are inactive while the 5′ regulatory region is bound by a repressor protein. Some genes or operons are regulated by more than one mechanism. For example, some bacterial genes and operons are subject to both a first, activation or derepression regulatory mechanism, and a second regulatory mechanism, called “catabolite repression.” Catabolite repression, also called “glucose catabolite repression” or “carbon catabolite repression,” is a phenomenon in which gene(s) under the control of a regulated promoter are also maintained in an unexpressed state until the concentration of glucose (the primary carbon source) falls below a threshold level, e.g., until conditions of glucose starvation. In other words, such a gene(s) cannot be expressed until two conditions are met: 1) glucose reduction/starvation and 2) activation or derepression of the regulated promoter. The occurrence of only one or the other condition is not sufficient to achieve expression of such gene(s). Among the genes and operons that have been found subject to catabolite repression are many that encode enzymes and/or pathways needed to utilize non-glucose carbon sources, i.e. alternative carbon sources.
The mechanism by which catabolite repression is effected is still undergoing intense scrutiny. In the case of some catabolite-repressed operons in E. coli, a transcriptional level of control has been assigned, in which catabolite repression is overcome by an “activatable promoter” mechanism. For example, the E. coli lactose operon (lacZYA) is maintained in an untranscribable state until glucose starvation permits a “catabolite activator protein” to bind to the operon's 5′ regulatory region; then, when lactose is present, Lac repressor protein is removed from a separate site(s) (the lac operator(s)) in the 5′ regulatory region, causing derepression, and transcription is initiated. Both conditions, i.e. both glucose starvation and the presence of lactose, are required for formation of lac operon-encoded mRNA in E. coli. 
In some cases, post-transcriptional controls are suspected. For example, there is evidence that, in Pseudomonads and closely related species, catabolite repression involving the crc gene is mediated post-transcriptionally. This is seen from studies of the regulation of bdkR [Ref. 7]. The bdkR protein, a transcriptional activator, is involved in the regulation of expression of branched-chain keto acid dehydrogenase in Pseudomonas putida. The data presented show that, in rich media, there is no bdkR protein detectable in wild type P. putida, despite the presence of bdkR transcripts. However, in a mutant P. putida in which crc is impaired or inactivated, bkdR protein is detected, bdkR transcript levels are slightly lower than those found in the wild type strain, and the transcript of the bdkR-regulated gene, bdkA, is induced about four-fold. Moreover, mutations identified in mutants in which the catabolite repression of bdkR is overcome, have been mapped to the crc gene, or to its cognate gene, vacB. In Shigella flexneri, the vacB protein regulates virulence genes post-transcriptionally; this presents additional, although circumstantial, evidence that crc acts post-transcriptionally [Ref. 13].
In commercial, prokaryotic systems, one of the key technological challenges associated with the production of proteins and chemicals by fermentation is total control of the transgene expression. The promoter selected for use in expressing the transgene of interest should have the following qualities. It should:                1. Separate growth from reaction;        2. Control gene expression for efficient/maximum product yield;        3. Induce the gene of interest at low/no cost; and        4. Allow no significant level of transcription in the repressed or non-induced state.        
For these reasons, regulated promoters are relied upon extensively. In particular, the lac promoter (i.e. the lacZ promoter) and its derivatives, especially the tac and trc promoters described in U.S. Pat. No. 4,551,433 to DeBoer, and the related promoters listed in
TABLE 1Commercial lac Promoters, Derivatives & RelativesCommercialBacterialPromoterInducerHost Cell(s)Reference(s)Ptac16IPTGE. coli,3, 4PseudomonadsPtac17IPTGE. coli,3PseudomonadsPlacUV5IPTGE. coli,3PseudomonadsPlacIPTGE. coli3, 4Plac(down)IPTGE. coli3T7IPTGE. coli,3, 4Pseudomonads
In a typical commercial, bacterial fermentation system, the host cell contains a construct in which a tac promoter is operably attached to a gene or operon whose expression is desired. The lacI gene, which is a constitutively expressed gene that encodes the Lac repressor protein which binds to the lac operator, is also included in the bacterial host cell (multiple copies of the lacI gene are usually included therein). After growth of a desired quantity or density of biomass, an inducer is added to the culture in order to derepress the tac promoter and permit expression of the desired gene or operon.
In commercial fermentation systems using a lac-type promoter, such as the tac promoter, the gratuitous inducer, IPTG (isopropyl-β-D-1-thiogalactopyranoside, also called “isopropylthiogalactoside”), is almost universally employed. However, IPTG is expensive and must be carefully controlled since it is significantly toxic to biological systems. Standard IPTG preparations are currently available at about US$18 per gram or about US$125 per 10 grams; these IPTG preparations also contain dioxane, which is likewise toxic to biological systems. Dioxane-free IPTG is also available on the market, but costs about twice the price of standard IPTG (i.e. currently about US$36 per gram or about US$250 per 10 grams). In addition to the problems of expense and high toxicity to the fermentation system itself, in situations in which the expression product or fermentation product is to be marketed, environmental and health regulatory issues arise in regard to the presence of IPTG therein, since IPTG also poses toxicity risks to humans, animals, and other biological organisms.
As a result, there is a need for promoters that are both useful for commercial fermentation production systems and activated by non- or low-toxicity inducers.
A further drawback of the use of lac promoters and their derivatives is that these promoters are “leaky” in that, even in a native, repressed state, the promoter permits a relatively high background level of expression. Therefore, multiple copies of the LacI repressor protein gene are usually included within the expression host cell in order to increase the degree of repression of the lac-type promoter. As a result, there is a need for promoters that are both useful for commercial fermentation systems and readily susceptible of being tightly controlled in an inactive state until induced.
In light of these concerns, several other, non-lac-type promoters have been proposed for controlling gene expression in commercial, prokaryotic fermentation systems (see Table 2).
TABLE 2Proposed Inducible Commercial non-lac PromotersBacterialPromoterInducerHost Cell(s)Reference(s)λPRHighE. coli,3, 4temperaturePseudomonadsλPLHighE. coli,3, 4temperaturePseudomonadsPmAlkyl- orPseudomonads4, 5halo-benzoatesPuAlkyl- orPseudomonads5halo-toluenesPsalSalicylatesPseudomonads5ParaArabinose inE. coli5the absenceof glucose
In regard to the first two promoters listed in Table 2, promoters induced by high temperatures are problematic: since high temperatures can be harmful to the host cell culture; since it is often impractical to generate an even temperature spike throughout the large-scale, commercial fermentation volume; and since it is preferred to operate commercial fermentation equipment at lower temperatures than required for such induction. The other four suggested promoters listed in Table 2 have, to the inventors' knowledge, not been demonstrated to function well in large-scale fermentation conditions; also, the alkyl- and halo-toluene inducers of the Pu promoter are significantly toxic to biological systems.
Thus, there remains a need for promoters that are useful for commercial fermentation production systems, activated by low-cost, low-toxicity chemical inducers, and tightly controlled.
In addition, in order to facilitate control of gene expression for production of proteins (and other expression products) and chemicals (processed by action of the expression products and/or the host cell) in a common fermentation platform using one prokaryotic organism, it is desirable to have a library of expression cassettes. These cassettes would each contain one or more of a variety of promoters that are of differing strengths, and/or induced under different growth conditions or by different chemicals. These expression cassettes would then be linked to various genes of interest to achieve total control of those genes under fermentation friendly conditions. The identification and optimization of a wide variety of growth-phase-dependent or chemically-inducible promoters is thus essential for control of (trans)gene expression during fermentation in such a fermentation platform.
Moreover, the construction of genetic circuits in which activation or induction of a first gene or operon leads to repression or activation of one or more subsequent genes or operons has been suggested as a means for very fine control of gene expression. Both linear (e.g., serial and cascade) and circular (e.g., daisy-chain) genetic circuits have been created. See, e.g., U.S. Patent Pub. No. 20010016354 A1 of Cebolla Ramirez et al. These genetic circuits require a number of different promoters in order to function, and, in commercial fermentation, genetic circuits would need to rely upon promoters that are effective in commercial fermentation conditions. Thus, there is a need in the field of genetic circuits for a greater variety of promoters useful in commercial fermentation.
As noted above, promoters for use in commercial fermentation systems should be tightly regulated so that expression occurs only upon induction, preferably effected late in the fermentation run. The chemicals used to induce the promoters must be low cost, low-toxicity to the host bacterium and other organisms, and must tightly regulate gene expression. In light of the above discussion, there is a need in the art for novel promoters that are tightly regulated and are induced at low cost using low-toxicity inducers.